Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies

The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.     

Genotoxicity of coke-oven and urban air particulate matter in in vitro acellular assays coupled with 32P-postlabeling and HPLC analysis of DNA adducts

This study is an in vitro part of the ongoing biomarker studies with population from a polluted region of Northern Bohemia and coke-oven workers from Czech and Slovak Republics. The aim of this study is to compare DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne particles. The crude extracts were fractionated into seven fractions by acid-base partitioning and silica gel column chromatography. In in vitro acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S9) and reductive activation mediated by xanthine oxidase (+XO) under anaerobic conditions. Both the butanol and nuclease P1 versions of 32P-postlabeling for detection of bulky aromatic and/or hydrophobic adducts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions similar for both the urban and coke-oven samples. The highest DNA adduct levels with S9-activation were detected for the neutral aromatic fraction, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detected for both the acidic fractions. Assuming additivity of compound activities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity in both in vitro assays (39 and 69%, +S9 and +XO, respectively). In contrast, the aromatic fraction constituting only 8% of total urban EOM mass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts for the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characterize some of the specific DNA adducts formed, we coupled TLC on 20x20 cm plates with HPLC analysis of 32P-postlabeled adducts. In both S9-treated samples of the aromatic fraction, we tentatively identified DNA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[+/-] (anti-BPDE), benzo[b,j,k]fluoranthenes (B[b]F, B[j]F, B[k]F), chrysene (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). These DNA adducts accounted for about 57% of total DNA adducts detected in both S9-treated samples of the aromatic fraction. DNA adducts of XO-treated samples were sensitive to nuclease P1 and HPLC profiles of the major adducts were markedly different from the major adducts of S9-treated samples. However, the combination of TLC and HPLC did not confirm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-nitroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detected by GC-MS in the slightly polar fraction. We concluded that the chemical fractionation procedure facilitates the assessing of DNA adduct forming ability of different chemical compound classes. However, based on the results obtained with the whole extracts, it does not fulfil a task of the actual contribution of individual fractions within the activity of the whole extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter.     

Detection of PCB adducts by the 32P-postlabeling technique

The purpose of this study was to determine whether lower chlorinated biphenyls would be bioactivated to electrophilic metabolites by microsomes alone or in combination with peroxidase. Monochloro- and dichlorobiphenyls were incubated with liver microsomes of rats treated with phenobarbital and beta-naphthoflavone, an NADPH-regenerating system, and deoxyguanosine 3'-monophosphate (dGp). The resultant adducts were analyzed by 32P-postlabeling either following microsomal incubation alone ("preoxidized") or coupled with subsequent oxidation with horseradish peroxidase/H2O ("oxidized"). The incubation of 4-monochlorobiphenyl (4-MCB) resulted in the formation of two minor adducts by microsomal activation alone. However, the oxidized sample showed two additional major adducts. Formation of the latter adducts was almost completely (> 80%) inhibited when the oxidation reaction was performed in the presence of ascorbic acid. The other test mono- and dichlorobiphenyls also formed 1-3 major adducts. Compared with microsomal activation alone, these adducts were enhanced after the oxidation reaction or detected only in the oxidized samples. These data suggest that (1) some adducts of the lower chlorinated biphenyls are derived from arene oxides and (2) many adducts may be formed by metabolism of the parent compounds to catechol and p-hydroquinone species, which are oxidized to semiquinones and/or quinones. The involvement of quinones and/or semiquinones was supported by UV/vis spectroscopic measurements, which showed that metabolites of 4-MCB can be oxidized to products with spectra characteristic of quinones. These data raise the possibility that lower chlorinated biphenyls may be genotoxic and may explain the fact that commercial polychlorinated biphenyl mixtures are complete rodent carcinogens.     

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 micrograms/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta 1 (TGF-beta 1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta 1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 micrograms/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta 1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.     

Covalent DNA adducts formed in mouse epidermis by benzo[g]chrysene

The metabolic activation in mouse skin of benzo[g]chrysene (B[g]C), a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar, was investigated. Male Parkes mice were treated topically with 0.5 micromol B[g]C and DNA was isolated from the treated areas of skin at various times after treatment and analysed by 32P-post-labelling. Seven major adduct spots were detected, at a maximum level of 6.55 fmol adducts/microg DNA. Mouse skin treated with the PAH benzo[c]phenanthrene (B[c]Ph) gave a total of 0.24 fmol adducts/microg DNA. B[g]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 micromol of the optically pure putative proximate carcinogens, the (+)- and (-)-trans benzo[g]chrysene-11,12-dihydrodiols, led to the formation of adducts which comigrated on TLC and HPLC with those formed in B[g]C-treated mice, which suggested that the detected adducts were formed by the fjord region B[g]C-11,12-dihydrodiol-13,14-epoxides (B[g]CDEs). To test this, the four optically pure synthetic B[g]CDEs were reacted in vitro with DNA and the heteroco-polymers poly(dA x dT) and poly(dG x dC) and these samples 32P-postlabelled. Co-chromatography, on both TLC and HPLC, of in vitro and in vivo adducts indicated that B[g]C is activated in mouse skin through formation of the (-)-anti-(11R,12S,l3S,14R) and (+)-syn-(11S,12R,13S,14R) B[g]CDEs. (-)-anti-B[g]CDE formed five adducts with DNA, two of them with adenine and three with guanine bases. (+)-syn-B[g]CDE formed one adduct with each of these bases in DNA. The adenine adducts accounted for 64% of the total major adducts formed in B[g]C-treated mouse skin. The route of metabolic activation or B[g]C is similar to that reported for B[c]Ph, but the extent of activation to the fjord region diol-epoxides is significantly greater in the case of B[g]C, as demonstrated by the higher levels of adduct formation in vivo.     

Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in mouse skin: their significance in tumor initiation

This paper reports expanded analyses of benzo[a]pyrene (BP)-DNA adducts formed in vitro by activation with horseradish peroxidase (HRP) or 3-methylcholanthrene-induced rat liver microsomes and in vivo in mouse skin. The adducts formed by BP are compared to those formed by BP-7,8-dihydrodiol and anti-BP diol epoxide (BPDE). First, activation of BP by HRP produced 61% depurinating adducts: 7-(benzo[a]pyrene-6-yl)guanine (BP-6-N7Gua), BP-6-C8Gua, BP-6-N7Ade, and the newly identified BP-6-N3Ade. As a standard, the last adduct was synthesized along with BP-6-N1Ade by electrochemical oxidation of BP in the presence of adenine. Second, identification and quantitation of BP-DNA adducts formed by microsomal activation of BP showed 68% depurinating adducts: BP-6-N7Ade, BP-6-N7Gua, BP-6-C8Gua, BPDE-10-N7Ade, and the newly detected BPDE-10-N7Gua. The stable adducts were mostly BPDE-10-N2dG (26%), with 6% unidentified. BPDE-10-N7Ade and BPDE-10-N7Gua were the depurinating adducts identified after microsomal activation of BP-7, 8-dihydrodiol or direct reaction of anti-BPDE with DNA. In both cases, the predominant adduct was BPDE-10-N2dG (90% and 96%, respectively). Third, when mouse skin was treated with BP for 4 h, 71% of the total adducts were the depurinating adducts BP-6-N7Gua, BP-6-C8Gua, BP-6-N7Ade, and small amounts of BPDE-10-N7Ade and BPDE-10-N7Gua. These newly detected depurinating diol epoxide adducts were found in larger amounts when mouse skin was treated with BP-7,8-dihydrodiol or anti-BPDE. The stable adduct BPDE-10-N2dG was predominant, especially with anti-BPDE. Comparison of the profiles of DNA adducts formed by BP, BP-7,8-dihydrodiol, and anti-BPDE with their carcinogenic potency indicates that tumor initiation correlates with the levels of depurinating adducts, but not with stable adducts. Furthermore, the levels of depurinating adducts of BP correlate with mutations in the Harvey-ras oncogene in DNA isolated from mouse skin papillomas initiated by this compound [Chakravarti et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426]. The depurinating adducts formed by BP in mouse skin appear to be the key adducts leading to tumor initiation.     

Characterization of DNA adducts formed by anti-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide

The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, an adduct in which the epoxide of the 11R, 12S, 13S, 14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[c]phenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[c]phenanthrene dihydrodiol epoxides react to much higher extents (approximately 4-fold) with DNA than did the benzo[g]chrysene derivative.     

32P-post-labelling analysis of DNA adducts formed in the upper gastrointestinal tissue of mice fed bracken extract or bracken spores

Bracken toxicity to both domestic and laboratory animals is well established and tumours are formed when rodents are treated with either bracken extracts or bracken spores. In this study we have administered bracken spores and extract to mice in order to investigate whether such exposure leads to the formation of DNA adducts. DNA, isolated from the upper gastrointestinal tract and liver, was digested to 3'-nucleotides. Adducts were extracted with butanol, 32P-post-labelled, separated by thin layer chromatography (TLC) and visualised and quantified using storage-phosphor technology. A cluster of adducts was clearly seen in the DNA of the upper gastrointestinal tract, but not liver, 5 and 24 h after treatment with bracken extract or bracken spores. These adducts were not observed in DNA extracted from vehicle-treated animals. Whereas, after 5 h adduct levels in extract and spore-treated animals were similar, after 24 h adduct levels in the extract-treated animals had diminished by > 75%, but levels in spore-treated animals remained similar to those found after 5 h. This suggests that the DNA-reactive compounds were being released slowly from the spores, even though the spores had been sonicated before administration. Adducts were also quantified after the addition of an internal standard (deoxyinosine 3'-monophosphate) by comparing the amount of label incorporated into the adducts with that found in a known amount of the internal standard. Adduct levels using this internal standard approach were similar to those found by direct measurement of radioactivity incorporated into the adduct, indicating that the labelling of adducts was quantitative. We have tried, unsuccessfully, to synthesise ptaquiloside, the principal carcinogenic component present within bracken. However, similar patterns of adducts were observed when two other compounds, (1-(4-chlorophenyl sulphonyl)-l-cyclopropane carbonitrile and 3-cyclopropylindeno [1,2-c] pyrazol-4-(O-methyl)oxime), which both contain a cyclopropyl ring, were administered to mice. The adducts detected in bracken-treated animals may, thus, have arisen from ptaquiloside but, whether these adducts arise directly from the compounds and bracken spores/extract themselves or via an indirect mechanism, remains to be determined. As bracken-induced DNA adducts are detectable in rodent tissues by a 32P-post-labelling procedure commonly employed to investigate DNA damage in human populations, it may prove possible to apply such approaches to determine human exposure.     

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling

OBJECTIVE: To determine the views of junior hospital doctors on their working conditions, NHS reforms and training, and to compare their views with those of consultants and managers. SUBJECTS: A questionnaire was distributed to 52 junior doctors, 19 consultants and 14 middle or senior grade managers in an acute NHS trust. CONCLUSIONS: Junior doctors had strong feelings about several areas covered in the questionnaire; in particular, more structured training without the requirement to undertake a higher degree would be welcomed. Shift systems are unpopular and the reduction of 'non-medical' tasks with a reduction in work intensity is perceived to be more important than further reductions in hours available for work.     

Monophosphate 32P-postlabeling assay of DNA adducts from 1,2:3,4-diepoxybutane, the most genotoxic metabolite of 1,3-butadiene: in vitro methodological studies and in vivo dosimetry

Among the main DNA-reactive metabolites of 1,3-butadiene (BD), both 1,2:3,4-butadiene diepoxide (BDE) and 1,2-epoxy-3-butene (BME) have been reported in mice and rats exposed to BD, but blood and tissue levels of these metabolites are much higher in mice than in rats under similar exposure conditions. BDE, being more reactive and genotoxic than BME, is thought to be responsible for the greater susceptibility of mice to BD carcinogenicity. While BDE is a DNA-alkylating agent and some BDE adducts have been characterized, no sufficiently sensitive method has been reported for studying BDE-DNA binding in vivo. In the present investigation, a modified dinucleotide/monophosphate version of the 32P-postlabeling assay was applied to detect BDE-DNA adducts, which were prepared by reacting BDE with calf thymus DNA or deoxyribooligonucleotides [(AC)10, (AG)10, (CCT)7 and (GGT)7] in vitro or with skin DNA of mice in vivo upon topical treatment. Optimal resolution by 2-D PEI-cellulose TLC of the highly polar 5'-monophosphate adducts was achieved at +4 degrees C using 0.3 M LiCI (DI) and 0.4 M NaCl, 0.04 M H3BO3, pH 7.6 (D2). The profiles of the 32P-postlabeled adducts were similar for calf thymus and skin DNA, with 3 major spots being detected. Adducts obtained in in vitro and in vivo experiments were compared by re- and cochromatography in 4 or 5 different solvents, and these experiments provided evidence that corresponding BDE adducts, for the most part, were identical and represented adenine derivatives. Guanine adducts were not detected by this method although literature data indicate their formation. Quantitatively, the assay responded linearly to adduct concentration, as shown in an experiment where BDE-modified skin DNA was serially diluted up to 81-fold with control DNA. The limit of detection was approximately 1 adduct in 10(8) normal nucleotides. Further, in an in vivo dosimetry study, skin DNA from groups of 8 individual mice treated with different doses of BDE (1.9, 5.7, 17, 51 and 153 mumol/mouse) for 3 days exhibited a linear relationship (r > or = 0.992) between adduct levels and dose. The results suggest that the 32P-postlabeling assay described herein will have utility in mechanistic studies and biomonitoring of DNA adduct formation from BDE and possibly other polar epoxides.     

DNA strand scission by benzo[a]pyrene diol epoxides

Syn-and anti-benzo[a]pyrene diol epoxides elicit a concentration-dependent nicking of superhelical Col E1 DNA in an in vitro reaction monitored by agarose gel electrophoresis and electron microscopy. This strand scission represents less than 1 percent of the DNA modification by diol epoxide. Kinetic analysis implicates the formation of unstable phosphotriesters, hydrolysis of which nick the DNA.     

Gas chromatographic-mass spectrometric determination of benzo[a]pyrene and chrysene diol epoxide globin adducts in humans

The efficacy of a newly developed gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) assay for measuring globin adducts of benzo[a]pyrene (B[a]P) and chrysene diol epoxides in human was evaluated. In this pilot study, smokers and nonsmokers were selected as exposed and nonexposed groups. Using [2H12]r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyren e ([2H12]trans,anti-B[a]P-tetraol) as an internal standard, B[a]P-tetraols released from globin after hydrolysis and derivatization were quantified by GC-NICI-MS-SIM. Levels of trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (chrysene-DE)-globin adducts were estimated by assuming that the recovery and the MS response of the perdeuterated B[a]P-tetraol internal standard reflected the recovery and MS response of chrysene tetraols. The assay was found to be reproducible and sensitive enough to detect both analytes in all samples. The mean levels of B[a]P-tetraols released from the corresponding benzo[a]pyrene diol epoxide (BPDE) globin adducts in smokers were significantly higher than those in nonsmokers, i.e., 2.6 +/- 0.6 SE fmol/mg globin (ranging from 1.2 to 7.8 fmol/mg globin) in smokers and 0.97 +/- 0.05 SE fmol/mg globin (ranging from 0.7 to 1.3 fmol/mg globin) in nonsmokers (P < 0.01). Interestingly, estimated levels of chrysene-DE-globin adducts in the same subjects were about two orders of magnitude higher than those of the globin adducts of BPDE. The mean of the chrysene-DE adducts in smokers was estimated to be 310 +/- 30 SE fmol/mg globin (ranging from 190 to 460 fmol/mg globin) and that in nonsmokers was 250 +/- 25 SE fmol/mg globin (ranging from 110 to 380 fmol/mg). Although the estimated mean of chrysene-DE adducts with globin in smokers appeared to be about 25% higher than in nonsmokers, the difference was not significant (P = 0.06). The results of this study demonstrate the feasibility of the GC-NICI-MS-SIM method for measurement of BPDE globin adducts in humans.     

Hemoglobin adducts of benzo[a]pyrene diolepoxide in newspaper vendors: association with traffic exhaust

Benzo[a]pyrene diol epoxide adducts with hemoglobin (Hb) were measured to detect human exposure to environmental benzo[a]pyrene from traffic exhaust. Benzo[a]pyrene tetrahydrotetrols (BPTs) released from Hb after acid hydrolysis were quantitated by gas chromatography-mass spectrometry after immunoaffinity chromatography. Fifty three newspaper vendors were enrolled. The median adduct concentration was 0.3 fmol BPTs/mg Hb in high density traffic-exposed vendors and < or = 0.1 fmol BPTs/mg Hb in those exposed to low density traffic; the difference was not significant (P = 0.09). Among non-smokers, adducts were detectable in 60% of high exposure subjects (median 0.3 fmol BPTs/mg Hb) and in 28% of those with low exposure (median < or = 0.1 fmol/mg Hb). This difference was significant (P = 0.02). In low exposure smokers the median of adducts was 0.26 fmol BPTs/mg Hb, while in low exposure non-smokers it was < or = 0.1 fmol BPTs/mg Hb (P = 0.08, not significant). Adduct concentration was no different for low and high density traffic-exposed smokers (P = 0.82). The data indicate a significant difference in adduct concentration related to traffic exhaust exposure among non-smokers.     

Mass spectrometric analysis of haemoglobin adducts formed by methyl bromide in vitro

An analytical procedure is described for the identification of the adducts formed by interaction of methyl bromide and haemoglobin. The reaction products of in vitro incubation of haemoglobin with methyl bromide have been characterised by electrospray mass spectrometry and gas chromatography-mass spectrometry. A prominent reactivity of several potential nucleophilic sites of haemoglobin was observed. Analogous results were recorded on blood samples of workers exposed to methyl bromide. The results obtained represent the basis for the complete structural characterisation of the modified haemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of alkylation degree and the identification of modified amino acids in proteins.     

Mass spectrometric sequencing of site-specific carcinogen-modified oligodeoxyribonucleotides containing bulky benzo[a]pyrene diol epoxide-deoxyguanosyl adducts

Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values.     

Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P-postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P-postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.     

Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.     

Mutagenicity of 6-sulfooxymethylbenzo[a]pyrene in Salmonella typhimurium and Chinese hamster V79 cells

STUDY DESIGN: To report on the preliminary results of preparing and reconstructing the pars interarticularis with a cable-screw construct. The success of previous techniques to repair the pars defect has been variable. OBJECTIVES: To assess the results of a new technique for stabilizing the pars interarticularis, using the strongest materials, pedicle screws, and cables in the strongest bony elements--the pedicle and lamina. SUMMARY OF BACKGROUND DATA: Previous techniques have been inadequate structurally to stabilize the pars interarticularis effectively, or the techniques were difficult to perform. Placing a screw across the defect was technically difficult and took away from the surface area of the fusion. The Scott technique used wiring between the transverse process and spinous process; and in the Morscher Technique, a hook screw was used to repair the pars defect--a technically difficult procedure, using bulky hardware. METHODS: Patients with pars interarticularis defects were carefully selected for this technique if they had primarily low back pain that did not respond to conservative treatment. The eligible patient had Grade I or less spondylolisthesis, little or no desiccation detectable on magnetic resonance imaging, and pain reproduced with injection of the pars defect. Surgical technique involved placing a special pedicle cable-screw into the pedicle of the involved vertebra. A double cable was passed underneath the lamina, threaded through the hole of the pedicle screw, and wrapped around the spinous process. The cables were simultaneously tensioned and crimped. A tricortical bone graft was compressed between the pedicle and lamina. RESULTS: Seven patients had this technique with a follow-up of 25.5 months (range, 19-37 months). The mean age was 20.5 years (range, 12-32 years), and the mean duration of symptoms was 31 months. All patients had severe pain before surgery that prevented participation in sports and normal daily activities. After surgery, results in five were rated as excellent and in two as good, according to the Prolo score. There were no failures of the cable-screw constructs, and all of the defects appear to have united solidly. CONCLUSION: The cable-screw construct uses the strongest anchors (the pedicle and the lamina) and uses compression obtained with cables to stabilize the pars interarticularis. Early results indicate that this is a safe and effective technique for this difficult problem.     

Sequence context profoundly influences the mutagenic potency of trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-purine nucleoside adducts in site-specific mutation studies

The postoligomerization method was used to prepare oligonucleotide 16-mers that contained dAdo or dGuo adducts, derived from trans opening of each enantiomer of the two diastereomeric benzo[a]pyrene 7,8-diol 9,10-epoxides, in two sequence contexts. These 16 oligonucleotides, along with the four corresponding oligonucleotides containing unsubstituted purines, were ligated into single-stranded DNA from bacteriophage M13mp7L2 and transfected into Escherichia coli SMH77. The mutagenic effects of replication past these adducts were then evaluated. The various adduct isomers induced point mutations at different frequencies and with different distributions of mutation types, as was anticipated. However, sequence context had the most substantial effects on mutation frequency. A high frequency of deletions of a single guanine was found in a context where the dGuo adduct was at the 3'-end of a run of five guanines, whereas no single base deletion was found in the other context studied, 5'-CGA-3'. Mutation frequencies in constructs containing dAdo adducts were much higher in a 5'-TAG-3' context (37-58%, depending on the individual isomer) than in a 5'-GAT-3' context (5-20%), and for a given adduct, mutation frequency was up to 10-fold higher in the former sequence than in the latter. These findings indicate that sequence context effects need more thorough evaluation if the goal of understanding the mechanism through which DNA adducts lead to mutation is to be achieved.